1. Field of the Invention
The present invention relates to (a) nucleic acid sequences which encode polypeptides having PhzO activity, that is, the ability to convert phenazine-1-carboxylic acid to 2-hydroxylated phenazines and (b) isolated polypeptides having this activity. The invention also relates to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in bacterial or plant cells to inhibit fungal pathogens.
2. Description of the Art
Root diseases caused by Gaeumannomyces graminis, Rhizoctonia, and Pythium and diseases caused by the pathogenic fungus Fusarium cause a significant adverse impact on the production of important crops worldwide. The root disease take-all, caused by Gaeumannomyces graminis var. tritici (Ggt), Rhizoctonia root rot, caused by Rhizoctonia solani and R. oryzae, and Pythium root rot caused by any of several Pythium species, notably, Pythium ultimum and P. irregulare, are important root diseases of small grain crops, e.g., wheat, barley, triticale, and rye, worldwide. Fusarium graminearum (Schwabe) and Fusarium culmorum are the primary causal agents of a disease known as Fusarium head blight, head blight, or scab, of wheat, barley, oats and rye. Fusarium solani causes root and crown rots, and Fusarium oxysporum causes wilts.
Widespread diseases of small grain crops and turf grass are caused by the soil-borne fungus Gaeumannomyces graminis (Gg), a member of the ascomycota class of fungi, and result in significant economic losses due to reductions in crop yield. Take-all, a disease caused by Gaeumannomyces graminis var. tritici (Ggt) occurs in all wheat-growing regions of the world and is probably the most important root disease of wheat and related small grains worldwide. Symptoms of wheat take-all include dark longitudinal lesions on roots; in severe cases, the entire root may become blackened with disease with the fungus migrating to in the crown of the wheat plant (where the crown roots originate) and the tillers (stems). Severely infected wheat plants are identified in the field by their white heads which result when infection of the crown by the fungus cuts off water transport to upper plant parts causing the plant to die prematurely. Yield losses can be considerable, up to 50% of the potential wheat yield. There are no resistant wheat cultivars, and registered fungicides perform inconsistently. Further, growers are being increasingly challenged to grow wheat with minimum or no tillage to reduce soil erosion. These practices increase the severity of take-all and other root diseases. Although wheat is particularly susceptible to the take-all fungus, many other Gramineae such as barley, rye, and triticale can also be infected.
Traditionally, take-all has been controlled by a combination of crop rotation and tillage, practices which reduce the inoculum potential of the pathogen. However, because long rotations are often not economically feasible and tillage contributes to soil erosion, the trend in cereal production is toward less tillage and two or three wheat crops before a break. Both of these practices exacerbate take-all. There is no known source of genetic resistance in wheat against take-all, and methods of chemical control are limited. The need for agriculture to become more sustainable and less dependent on chemical pesticides has necessitated the development of alternative approaches to control take-all and other soil-borne diseases.
Other Gg fungi, for example, Gaeumannomyces graminis var. avenae (Gga), infect oats and grasses and have been identified as causing take-all patch in turf grasses such as bent grass. Gaeumannomyces graminis var. graminis (Ggg) infects some grasses and has been suggested as causing crown sheath rot in rice.
Rhizoctonia, a member of the basidiomycota class of fungi, causes root and stem rot on most food, fiber, and ornamental plants throughout the world, including small grain crops, turf grass, asparagus, canola, corn, sugarbeet, tomatoes, potatoes, peas, rice, beans, soybeans, strawberries, zucchini, and cotton. Root rot on small grain crops caused by Rhizoctonia occurs throughout the United States Pacific Northwest, in Australia, and South Africa, and potentially throughout the temperate regions of the world wherever small grains are grown, especially if grown with reduced or no-tillage (direct drilling). Rhizoctonia root rot caused by R. solani AG8 begins as brown cankerous lesions on the seminal and crown roots that eventually girdles and then severs the roots. Plants with roots pruned off by this disease remain stunted and eventually die without making heads. The disease tends to affect plants in patches and has given rise to other names, such as bare patch disease, purple patch, crater disease, and barley stunt disorder. Of all small grain crops, barley is especially susceptible to R. solani AG8. Rhizoctonia oryzae infects the embryos of germinating seeds, preventing germination or limiting the formation of seminal roots to only one or two when healthy seedlings produce five or six seminal roots. These two Rhizoctonia species, together with Rhizoctonia cerealis and possibly other Rhizoctonia species occur as different mixtures, depending on the soil, cropping systems, weed management practices, and possibly other factors not yet identified.
The soil-borne pathogen complex of Pythium spp. comprises a group of fungi that are among the most successful of all microbial colonists in agricultural soils. It is estimated that nearly all cultivated soil in the world contains spores of at least one, two, three, and even as high as ten Pythium species. Pythium, a member of the oomycetes class of fungi, like Rhizoctonia, affects virtually all food, fiber, and ornamental plants throughout the world. Examples of these plants are given above. Pythium damage to small grains begins as embryo infections and associated poor emergence or stand establishment and continues as destruction of the fine lateral rootlets and root hairs. Plants with Pythium root rot have the appearance of plants without enough fertilizer, because the disease limits the absorptive capacity of the root system through destruction of fine rootlets and root hairs. There are several species of Pythium with ability to attack cereals, either embryos of germinating seeds, root tips and fine rootlets, or all of these delicate and usually juvenile or meristematic tissues.
Fusarium head blight or scab is a fungal disease of wheat, barley, oats, rye, and wheatgrasses that affects both grain yield and quality. It occurs worldwide, particularly when temperatures and humidity favor the proliferation of the causal agent, Fusarium graminearum, at the time of heading. Head blight has caused losses in the billions of dollars to United States and Canadian growers and processors within this decade. Yield and grain quality losses of wheat due to Fusarium head blight approached one billion dollars in Minnesota, North Dakota, and South Dakota in 1993 and 200-400 million dollars across the region in subsequent years. Losses were in excess of 300 million dollars in Ohio, Michigan, Indiana, and Illinois in 1995 and 1996. Quality of grain is also compromised since infected grain is usually contaminated with a mycotoxin, vomitoxin or DON, produced by the fungus that is detrimental to humans and livestock. In addition, the disease has threatened barley production in the upper Midwest because brewers have imposed zero tolerance limits for vomitoxin in grain.
Certain strains of root-colonizing fluorescent Pseudomonas spp. have gained attention in recent years because they produce broad-spectrum antibiotic metabolites that can provide protection against various soilborne root pathogens (Thomashow and Weller, pp. 187-235. In G. Stacey and N. T. Keen (ed.), Plant-Microbe Interactions. Chapman and Hall, New York, N.Y., 1996). One such class of antibiotics, the phenazines, encompasses a large family of heterocyclic nitrogen-containing compounds produced in late exponential and stationary phase. The ability to produce phenazines is limited almost exclusively to bacteria, and has been reported in members of the genera Pseudomonas, Streptomyces, Nocardia, Sorangium, Brevibacterium, and Burkholderia (Turner and Messenger, Advances in Microbial Physiol. 27: 211-275, 1986). There are currently over 50 known phenazine compounds with the same basic structure, differing only in the derivatization of the heterocyclic core. These modifications largely determine the physical properties of phenazines and influence their biological activity against plant and animal pathogens.
The broad-spectrum activity exhibited by phenazine compounds against fungi and other bacteria is not understood. It is thought that they can diffuse across the membrane and, once inside the cell, accept a single electron, disrupting respiration by interfering with the normal process of electron transport. This results in the overproduction of O2xe2x88x92 and H2O2, which overwhelm cellular superoxide dismutases and ultimately cause cell death. The cellular superoxide dismutases in P. aeruginosa, which produces the phenazine compound pyocyanin, are more active than those of phenazine-non-producing bacteria such as Escherichia coli, and they provide protection against phenazines (Hassan and Fridovich, J. Bacteriol. 141:156-163, 1980; Hassett et at., J. Bacteriol. 177:6330-6337, 1995).
Several studies conducted in the early 1970s revealed tight links between phenazine biosynthesis and the shikimic acid pathway (Turner and Messenger, 1986, supra), but the biochemistry and genetic control of phenazine synthesis are still not fully understood. Chorismic acid has long been recognized as the branchpoint from the shikimic acid pathway to phenazine synthesis (Longley et al., Can. J. Microbiol. 18:1357-1368, 1972). Studies with radiolabeled precursors suggest that the phenazine core is formed by the symmetrical condensation of two molecules of chorismic acid (Chang et al., Can. J. Microbiol. 72:581-583, 1969; Herbert et al., Tetrahedron Letters 8:639-642, 1976; Hollstein and McCamey, J. Org. Chem. 38:3415-3417, 1973; Longley et al., 1972, supra), while the amide nitrogen of glutamine serves as the immediate source of nitrogen in the heterocyclic nucleus of phenazine compounds (Rxc3x6mer and Herbert, Z. Naturforsch. C 37:1070-1074, 1982). Phenazine-1,6-dicarboxylic acid is the first phenazine formed, and it is thought to be converted to phenazine-1-carboxylic acid (PCA), a key intermediate in the synthesis of other phenazines by fluorescent pseudomonads (Bying and Turner, J. Gen. Microbiol. 97:57-62, 1976; Herbert et al., 1976, supra; Hollstein and McCamey, 1973, supra; Messenger and Turner, FEMS Microbiol. Lett. 18:65-68, 1983).
Genetic studies in fluorescent Pseudomonas spp., the only microorganisms for which the genes responsible for the assembly of the heterocyclic phenazine nucleus have been cloned and sequenced, support this model. The phenazine biosynthetic loci from P. fluorescens 2-79 (Mavrodi et al., J. Bacteriol. 180:2541-2548, 1998), P. aureofaciens [synonym: P. chlororaphis] 30-84 (Mavrodi et al., 1998, supra; Pierson III et al., FEMS Microbiol. Lett. 143:299-307, 1995), P. aeruginosa PA01 (D. V. Mavrodi and L. S. Thomashow, unpublished), and P. chlororaphis PCL1391(Chin-A-Woeng et al., Pseudomonas ""99: Biotechnology and Pathogenesis, S48, Maui, Hi., 1999) are highly conserved. Each contains a seven-gene core operon regulated in a cell-density dependent manner by homologues of LuxI and LuxR (D. V. Mavrodi and S. K. Farrand, unpublished; Latifi et al., Mol. Microbiol. 17:333-343, 1995; Wood and Pierson III, Gene 108:49-53, 1996). In P. fluorescens 2-79, P. aureofaciens 30-84, and P. chlororaphis PCL1391, the phzI/R genes are found directly upstream from the phenazine core. Phenazine production in Pseudomonas aeruginosa is controlled by two sets of regulatory proteins, rhlI/R and lasI/R, located elsewhere in the genome. The core gene products PhzC, PhzD, and PhzE, which are homologous with PhzF, PhzA, and PhzB in strain 30-84, are similar to enzymes of shikimic and chorismic acid metabolism. Sequence comparisons of PhzD and PhzE with other chorismate-modifying enzymes have shed new light on probable intermediates in the PCA pathway, suggesting that phenazine synthesis proceeds via the intermediates aminodeoxyisochorismic acid and 3-hydroxyanthranilate (Mavrodi et al., 1998, supra) rather than anthranilate, as suggested previously (Essar et al., J. Bacteriol. 172:884-900, 1990). It should be noted that P. aureofaciens strains are also known as P. chlororaphis. In cases where the cited references use the term P. aureofaciens, this designation has been retained.
Although the phenazine biosynthetic loci of fluorescent pseudomonads are highly homologous, individual species typically differ in the range of phenazine compounds they produce. Previous work by Pierson et al., 1995, supra, suggested that the phzC gene of P. aureofaciens 30-84, and in particular the last 28 amino acids of the PhzC protein, are essential for the production 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine (2-OH-PHZ), derivatives characteristic of strains previously designated P. aureofaciens but now classified as P. chlororaphis (Johnson and Palleroni, Inter. J. Syst. Bacteriol. 39:230-235, 1989).
Phenazine product specificity directed to compounds having biocontrol activity or active in the biosynthesis of phenazines having biocontrol activity and transformed microorganisms and plants producing such compounds can provide an important resource for control of plant disease or lead to the development of novel pharmaceutical products.
The present invention is directed to phzO nucleic acid sequences which encode polypeptides having PhzO activity, that is, the ability to convert phenazine-1-carboxylic acid to a 2-hydroxylated phenazine. The invention is further directed to isolated polypeptides having PhzO activity. The invention also relates to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in bacterial or plant cells to inhibit fungal pathogens.
More particularly, the invention encompasses isolated nucleic acid molecules that encode PhzO polypeptides. The phzO gene sequence is specifically exemplified herein. As discussed in detail below, the novel gene designated phzO was located 271 nucleotides downstream from phzD, in the core phenazine operon phzXYFABCD of P. aureofaciens 30-84, and was preceded by a well-conserved potential ribosome binding site, GAGG. phzO encoded a 491-amino acid protein with a calculated molecular mass of 55.1 kDa. Homology searches with the deduced amino acid sequence revealed similarity to bacterial aromatic hydroxylases and monooxygenases (Table 3). Phylogenetic analysis of these aligned protein sequences resulted in the tree shown in FIG. 3. The high bootstrap values (from 1,000 resamplings) showed the robustness of these groups.
As shown in detail, below, results of our study show clearly that phzXYFABCD, the core phenazine biosynthetic operon of strain 30-84, is responsible only for the synthesis of phenazinc-1-carboxylic acid (PCA). When transformed with these genes, the sole phenazine product synthesized by P. fluorescens strains Q8r1-96 and M4-80R (which themselves do not produce phenazines) was phenazine-1-carboxylic acid. However, phenazine-non-producing strains transformed with phzXYFABCD and phzO, or P. fluorescens 2-79 transformed with phzO, synthesized hydroxyphenazine compounds in addition to phenazine-1-carboxylic acid, and E. coli expressing phzO rapidly converted exogenously-supplied phenazine-1-carboxylic acid to 2-hydroxylated phenazine products. Finally, a mutant of 30-84 inactivated in phzO produced only phenazine-1-carboxylic acid.
Nucleic acid sequences which hybridize specifically to the coding sequence or its complement under medium or high stringency conditions and which encode a polypeptide having PhzO activity are also encompassed by the present invention.
Nucleic acid sequences having at least 50% sequence identity with the exemplified phzO sequence as described in detail, below, and which encode a polypeptide having PhzO activity are also encompassed by the present invention.
The present invention is also directed to isolated polypeptides having PhzO activity. A polypeptide having an amino acid sequence which has at least 60% sequence identity with the exemplified PhzO polypeptide as described in detail, below, is encompassed by the invention. Polypeptides encoded by a nucleic acid sequence which hybridizes under medium or high stringency conditions with exemplified nucleic acid sequences as discussed in detail, below, are also encompassed by the invention. Variants of the polypeptides are encompassed by the invention as well as fragments having PhzO activity.
A further aspect of the invention is the provision of recombinant nucleic acid molecules containing the sequences encoding polypeptides having PhzO activity. Such molecules include, for example, recombinant vectors, such as cloning, expression or transformation vectors, which contain a DNA sequence encoding a polypeptide having PhzO activity.
Another aspect of the invention is the provision of cells which are transformed by the above vectors or DNA sequences. A particular use of the invention is the provision of bacterial cells transformed with a nucleic acid sequence encoding a polypeptide having PhzO activity to provide bacterial strains which produce PhzO.
In one aspect of the invention, microorganism strains which produce phenazines such as phenazine-1-carboxylic acid are transformed with a nucleic acid sequence which encodes for a polypeptide having PhzO activity, to thereby obtain transformed strains which produce a 2-hydroxylated phenazine or which produce increased amounts of a 2-hydroxylated phenazine.
In another aspect of the invention, phenazine non-producing microorganism strains are transformed with a phz operon capable of producing phenazine-1-carboxylic acid and a nucleic acid sequence which encodes for a polypeptide having PhzO activity, to thereby obtain transformed strains which produce a 2-hydroxylated phenazine.
In another aspect of the invention, microorganism strains which contain a nucleic acid sequence which encodes for a polypeptide having PhzO activity are fed an exogenous phenazine compound such as phenazine-1-carboxylic acid to produce a 2-hydroxylated phenazine.
The invention is also directed to biologically pure cultures of the microorganism strains described above. A particular use of the strains is to suppress (inhibit the incidence of or reduce the incidence or severity of) or control fungal diseases, for example, soilborne plant root diseases.
A further aspect of the invention is application of the strains of the invention or compositions comprising the strains. For example, the strains or compositions comprising the strains can be used as a seed, soil, furrow treatment or drench, for application to suppress or control fungal disease.
Another aspect of the invention is the provision of plant cells transformed with a nucleic acid sequence encoding a polypeptide having PhzO activity to provide transformed plants which produce PhzO, to provide plants having resistance or enhanced resistance to fungal diseases. In one embodiment, plants containing a nucleic acid sequence which encodes for a polypeptide having PhzO activity are supplied with an exogenous source of phenazine-1-carboxylic acid (of chemical or microbial origin) to produce a 2-hydroxylated phenazine.
A further aspect of the invention is the provision of oligonucleotide probes capable of detecting a phzO gene or functional equivalents thereof, and the use of the probes to isolate nucleic acid sequences encoding a phzO gene or functional equivalent thereof. The nucleic acid sequences which specifically hybridize to the probes and which encode a functional polypeptide having PhzO activity are encompassed by the present invention.
Using the nucleic acid sequences of the invention facilitates the isolation of homologous genes from microorganisms to obtain genes which protect host cells, including bacteria, and plants against fungal pathogens.
The invention is also directed to methods of producing and using the polypeptides of the invention, and to methods of producing 2-hydroxylated phenazines.